Cloning,expression, and biochemical characterization of 3-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-<Emphasis Type="Italic">manno</Emphasis>-2-octulosonate-8-phosphate (KDO8P) synthase from the hyperthermophilic bacterium<Emphasis Type="Italic"> Aquifex pyrophilus</Emphasis>

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by ¹H NMR analysis. KDO8P synthase showed maximum activity at 80 °C and pH 5.5–6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 °C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 °C were K_m^A5P=70 M, K_m^PEP=290 M, and k_cat=4 s^–1. The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.Communicated by G. Antranikian     

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Discovery of nigerose phosphorylase from Clostridium phytofermentans

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease. Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999     

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083^T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate. Received: 15 March 1999 / Received revision: 8 June 1999 / Accepted: 11 June 1999     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g · ml⁻¹, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. Received: 24 September 1997 / Accepted: 12 November 1997     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

¹⁴C-Labelled octulose phosphates were formed during photosynthetic ¹⁴CO₂ fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero- d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though ¹⁴C-labelled d-glycero- d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. ¹⁴CO₂ predominantly labelled positions 5 and 6 of d-glycero- d-ido-octulose 1,8-P₂ consistent with labelling predictions of the modified scheme. The kinetics of ¹⁴CO₂ incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to ¹²CO₂ rapidly displaced the ¹⁴C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO₂.) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO₂ assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO₂ fixation at rates in excess of 120 μmol h⁻¹ mg⁻¹ Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero- d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that ¹⁴C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO₂ fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Modulation of gene expression by (CA)n microsatellites in the filamentous ascomycete Podospora anserina

A microsatellite consisting of the alternating pyrimidine-purine sequence (CA)_n.(TG)_n is found to occur in very conserved form in the genome of various races of the filamentous ascomycete Podospora anserina. Screening of a cDNA library revealed that this sequence is frequently transcribed. In this study, we focused our attention on a short (CA)₅ microsatellite located in the 5′ untranslated sequence of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of P. anserina. Specifically, we investigated whether or not the number of repeat units present in the microsatellite affects the expression of the β-d-glucuronidase (gusA) reporter gene introduced on an autonomously replicating plasmid into fungal protoplasts. The results show that an increase in the number of microsatellite repeat units positively affects reporter gene expression. Received: 27 November 1998 / Received revision: 12 February 1999 / Accepted: 20 March 1999     

Glycerol andl-α-Glycerol-3-phosphate uptake bypseudomonas aeruginosa

Uptake activities for both glycerol andl-α-glycerol-3-phosphate inPseudomonas aeruginosa strain PAO were induced during growth in the presence of either glycerol ordl-α-glycerol-3-phosphate. Succinate, malate, and glucose exerted catabolite repression control over induction of both uptake activities. Glycerol uptake exhibited saturation kinetics with an apparentK _m of 13 μM and aV _max of 73 nmol/min/mg cell protein. The uptake ofl-α-glycerol-3-phosphate was inhibited by the presence of glycerol, but uptake of glycerol was unaffected by exogenousl-α-glycerol-3-phosphate. Uptake of both substrates by starved, induced cells was stimulated by exogenously providedd-glucose, 2-deoxy-d-glucose,d-gluconate, orl-malate. In a mutant deficient in gluconate uptake and glucose dehydrogenase (EC 1.1.1.47) activities,d-glucose, 2-deoxy-d-glucose, andd-gluconate exerted little or no effect on the uptake of either substrate, butl-malate markedly stimulated the processes. The uptake of both glycerol andl-α-glycerol-3-phosphate, by either starved or unstarved cells, was inhibited by a number of metabolic poisons, including arsenate, azide, cyanide, 2,4-dinitrophenol, and iodoacetate.     

The stable phylogenetic distribution of the recently evolvedl-phenylalanine-inhibited isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in enteric bacteria

Escherichia coli and some other enteric bacteria possess three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, each of which is inhibited by one aromatic amino acid. Thel-phenylalanine-sensitive isozyme of DAHP synthase has evolved most recently since it is absent in all other members of the Gram-negative cluster that contains enteric bacteria as a subcluster. A comprehensive survey of enteric genera was carried out to determine whether the newly evolved isozyme is a stable, conserved trait. The results obtained show that all the genera of the contemporaryEnterobacteriaceae family possess the recently evolved phenylalanine-sensitive isozyme in addition to the tyrosine-and tryptophansensitive isozymes of DAHP synthase. However, physiological manipulation was usually necessary to derepress the tryptophan-sensitive DAHP synthase in order to demonstrate its presence.Florida Experiment Station Journal Series No. 9603.